The Cell–Cell Communication Signal Indole Controls the Physiology and Interspecies Communication of Acinetobacter baumannii

ABSTRACT Many bacteria utilize quorum sensing (QS) to control group behavior in a cell density-dependent manner. Previous studies have demonstrated that Acinetobacter baumannii employs an N-acyl-L-homoserine lactone (AHL)-based QS system to control biological functions and virulence. Here, we report that indole controls biological functions, virulence and AHL signal production in A. baumannii. The biosynthesis of indole is performed by A1S_3160 (AbiS, Acinetobacter baumannii indole synthase), which is a novel indole synthase annotated as an alpha/beta hydrolase in A. baumannii. Heterologous expression of AbiS in an Escherichia coli indole-deficient mutant also rescued the production of indole by using a distinct biosynthetic pathway from the tryptophanase TnaA, which produces indole directly from tryptophan in E. coli. Moreover, we revealed that indole from A. baumannii reduced the competitive fitness of Pseudomonas aeruginosa by inhibiting its QS systems and type III secretion system (T3SS). As A. baumannii and P. aeruginosa usually coexist in human lungs, our results suggest the crucial roles of indole in both the bacterial physiology and interspecies communication. IMPORTANCE Acinetobacter baumannii is an important human opportunistic pathogen that usually causes high morbidity and mortality. It employs the N-acyl-L-homoserine lactone (AHL)-type quorum sensing (QS) system, AbaI/AbaR, to regulate biological functions and virulence. In this study, we found that A. baumannii utilizes another QS signal, indole, to modulate biological functions and virulence. It was further revealed that indole positively controls the production of AHL signals by regulating abaI expression at the transcriptional levels. Furthermore, indole represses the QS systems and type III secretion system (T3SS) of P. aeruginosa and enhances the competitive ability of A. baumannii. Together, our work describes a QS signaling network where a pathogen uses to control the bacterial physiology and pathogenesis, and the competitive ability in microbial community.

9. l 78 and l 238: consider adding ref that first showed indole is an inter-species signal by tightening epithelial junctions (www.pnas.org/cgi/doi/10.1073/pnas.0906112107 ), which has led to many later ones showing indole is important for mood, skin, liver, brain development, aging, etc. Far better to use the primary reference rather than the over-reliance on the indole review articles. 13. l 56 SI: should be "qRT-PCR" and should refer to turbidity, not absorbance (i.e., no OD) for cell growth. Authors report indole production by AbiS gene from Acinetobacter baumannii and its roles in A. baumannii and against Pseudomonas aeruginosa. While the study is generally interested, there are several serious issues that should be further addressed.
Major comment 1: How much extracellular indole was found in the culture supernatant of A. baumannii? If Fig. 3 is correct, the wild type of A. baumannii produces only 400 nM (0.4 uM). It is very small amount compared to other indole-producing strains of E. coli and Vibrio strains that often produces more than 400 uM (1000 times more than this study). If so, other assays with indole addition (50 and 100 uM) is not physiological relevant. For the study of indole biosynthesis by AbiS in E. coli, indole deficient E. coli (tnaA mutant) should be used, for example, Fig. 4A. Then, authors can clearly conclude that indole biosynthesis pathway by AbiS is different from that by tnaA.
Major comment 2: While the identification of AbiS gene in this study is novel, the competition assay with indole against P. aeruginosa is not new. A key previous study (Indole Production Promotes Escherichia coli Mixed-Culture Growth with Pseudomonas aeruginosa by Inhibiting Quorum Signaling, Applied and Environmental Microbiology, 2012, 78, https://doi.org/10.1128/AEM.06396-11). The previous study already reported that indole from E. coli inhibited pyocyanin production and other AHL-regulated virulence factors in P. aeruginosa, which is similar to Fig. 2 and Fig. 7. Please compare current results to previous results carefully. Also, it has been reported that indole enhanced the competitive ability against other bacteria, such as Staphylococcus aureus, Candida albicans, Paenibacillus alvei, Serratia marcescens, etc, which are worthy to mention.
Major comment 3: Authors insisted that tryptophan is not a precursor for the biosynthesis of indole by AbiS. Then what is the indole precursor? Also, authors said that the indole biosynthetic pathway performed by AbiS in A. baumannii is different from that in E. coli. Then, how A. baumannii can produce indole? Also, it has been mentioned that homologs of AbiS were found in Acidovorax, Acinetobacter, Alcanivorax, Marinobacter and Noviherbaspirillum (Table S3). Then, can these bacteria produce indole?
Reviewer #3 (Comments for the Author): This manuscript describes a new indole/AbiS system in A. baumanni with multiple signalling effects, which would be interesting to a wide range of audience. However, further information and experimental details are needed to confirm the novelty of this system.

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Point-to-point response to reviewers' suggestions
Reviewer comments: Reviewer #1 (Comments for the Author): The authors indicate that indole increases A. b. virulence by increasing AHL synthesis, confirm indole reduces P. a. virulence (as indicated by ref. 32), and claim indole is produced by an enzyme different from tryptophanase in A. b. The manuscript is well-written. "*" indicates major problems below which need to be addressed. One of the main issues is the lack of solvent (negative controls) throughout; this is critical given the mistakes in the indole literature related to the use of solvents as indole diluents (DMSO here, which has significant toxicity, if not used less than 0.2 vol%). Also, the constructed strains must be validated, and AbiS was not shown to synthesize indole (it was shown genetically to influence indole concentrations).
Response: Thanks a lot for your good comments and suggestions. In our experiments, we have already added the same amount of solvent used for the compounds as a control, we have added this information in the Figure Legend in the revised manuscript. All constructed strains were validated by DNA sequencing. In addition, we found that AbiS was not able to synthesize indole from tryptophan, but it can synthesize indole from other precursors inside cells. *1. Title and l 27, 41: replace "modulates" with a more informative word; i.e., indicate how indole affects physiology (increases or decreases virulence etc.). Avoid vague writing.
Response: We have changed "modulates" to "controls" and "increases" as suggested.
2. l 33: indicate more specifically how indole "interfered with its QS" for P. a. since it is already published that indole reduces P. a. virulence (current ref 32). Replace "interfering" with a more informative word.
Response: (Line 33) We have changed this sentence to "Moreover, we revealed that indole from A. baumannii reduced the competitive fitness of Pseudomonas aeruginosa by inhibiting its QS systems and type III secretion system (T3SS)."

l 39: add ref for first sentence.
Response: There is no citation allowed in "Importance" section, but we have cited the relevant reference in the "Introduction" section. 4. l 39: delete "It was demonstrated that" here and anywhere else the ms.  13. l 56 SI: should be "qRT-PCR" and should refer to turbidity, not absorbance (i.e., no OD) for cell growth.
Response: It has been changed to "Quantitative RT-PCR analysis" and "2 x 10 9 cfu/mL". Response: We can conclude that AbiS is involved in the biosynthesis of indole in Acinetobacter baumannii, but we have not identified its precursor. Till now, we found that AbiS is different from TnaA (Fig 5b, 5c). Response: (Line 250) It has been changed to: "We also found that deletion of abiS caused an approximately 37% reduction in mCherry fluorescence intensity when the cells were cocultured with PAO1 at 48 h post-inoculation compared with the A. baumannii wild-type strain (Fig. 8b)." 26. l 235: should be "behavior".
Response: (Line 258) It has been changed to: "In microbial communities, many bacteria use Response: Reference has been added in the position as suggested as Reference 17.

Reviewer #2 (Comments for the Author):
Authors report indole production by AbiS gene from Acinetobacter baumannii and its roles in A.
baumannii and against Pseudomonas aeruginosa. While the study is generally interested, there are several serious issues that should be further addressed.
Major comment 1: How much extracellular indole was found in the culture supernatant of A.
baumannii? If Fig. 3  aeruginosa, which is similar to Fig. 2 and Fig. 7 baumannii can produce indole? Also, it has been mentioned that homologs of AbiS were found in Acidovorax, Acinetobacter, Alcanivorax, Marinobacter and Noviherbaspirillum (Table S3).
Then, can these bacteria produce indole?
Response: Thanks a lot for your good suggestions. We have found that tryptophan is not a precursor for the biosynthesis of indole performed by AbiS (Fig. 5b, 5c), but we don't know what is the precursor, which is worth to being investigated in the future. We will also measure the production of indole in other bacterial species in the next project.
Reviewer #3 (Comments for the Author): This manuscript describes a new indole/AbiS system in A. baumanni with multiple signalling effects, which would be interesting to a wide range of audience. However, further information and experimental details are needed to confirm the novelty of this system.

The manuscript describes a new indole synthesis pathway in A. baumannii via A1S_3160
protein (named AbiS by the authors). Also, the authors report two novel aspects of indole signalling in A. baumannii, including (i) physiological effects on biofilm formation, motility and virulence via modulating AHL QS system, and (ii) interspecies competitive effect against the non-indole producer P. aeruginosa via repressing its QS system & T3SS.
The manuscript is well written and clearly structured but there are few general and experimental issues that need to be addressed by the authors. One major issue is related to the authors suggestion that the indole biosynthetic pathway performed by AbiS in A. baumannii is different from that of TnaA in E. coli. The authors suggest that tryptophan is not a precursor for indole production but provide no clear evidence to support their finding. Can indole be produced by A.
baumannii in a minimal media lacking tryptophan? Can indole be produced in an E. coli ΔtnaA (abiS) growing in a minimal media lacking tryptophan? If so, this should support the authors' suggested mechanism.
Provided that tryptophan is not the precursor for indole synthesis via AbiS, the authors did not provide details on the alternative precursor?
Response: Thanks a lot for your good suggestions. We have constructed E. coli tnaA deletion mutant and grown these strains in 0.5 x LB and a minimal media lacking tryptophan as suggested. As shown in Fig. 5, exogenous addition of tryptophan will not affect the production of indole performed by AbiS, but will significantly induce the indole production performed by TnaA.
In addition, △tnaA (abiS) produces almost the same amount of indole in the 0.5 x LB and tryptophan deficient minimal media, suggesting that tryptophan will not affect the biosynthesis of indole via AbiS.
Other concerns are summarised below.
Lines 61: CpxA has been recently proposed as an indole sensor in E. coli. However, two other envelope stress response modules (BaeSR and the phage shock pathway) respond to indole.
Therefore, there has been suggestions that the cell membrane might be responsible for and show that indole can be produced again by complementing with either tnaA or abiS. This would be the only way to confirm that abiS is working in E. coli.
Response: Thanks a lot for your good suggestions. We have revised these data in Fig. 5 as suggested.
Line 165 -166 & Figure 4b: Assuming that the authors are using LB for this experiment, the supernatant concentration of indole in an E. coli culture would be between 300 -500 µM (LB has around 300 -500 µM tryptophan and the conversion rate of tryptophan to indole via TnaA is 1:1).
Thus, using 100 µM tryptophan for supplementation is a strange choice. The authors should comment on their choice! Response: (Line 178-195) Thanks a lot for your good suggestions. We have revised these experiments and results as suggested (Fig. 5).
Line 166 -168: 100 μM tryptophan is unlikely to be physiologically relevant therefore it is not surprising that the authors found no effect on A. baumannii. This does not rule out that tryptophan might be a precursor for indole production via abiS. The authors should supplement with tryptophan in the range of 300 -500 µM.
Response: Thanks a lot for your good suggestions. As previous study showed that indole production by the TnaA in E. coli was determined by exogenous tryptophan (Li et al., 2013, Microbiology (Reading) 159:402-410). As LB culture medium contains 0.5-0.6 mM tryptophan, we then cultured the strains in 0.5 × LB medium and found that exogenous addition of 300 uM tryptophan could not affect the production of indole in the ∆tnaA(abiS) strain, but will significantly induce the production of indole in ∆tnaA(tnaA), suggesting that tryptophan is not a precursor for the biosynthesis of indole by AbiS. Response: Thanks a lot for your good suggestions, we have revised this mistake as suggested.  Thank you for submitting your manuscript to Microbiology Spectrum. When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed instructions on submitting your revised paper are below.

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